Throughout this application various publications are referred to by partial citations within parenthesis. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications, in their entireties, are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Primary amino acid sequence and signal transduction data are now published for four cloned 5-HT1-like receptors, three cloned 5-HT2 receptors, and one 5-HT3 receptor. Analysis of the sequence homology as well as the signal transduction pathways of these receptors leads to their grouping on the basis of these attributes: The 5-HT1 subfamily includes: 5-HT1A (Kobilka et al., 1987; Fargin et al., 1988), 5-HT1B/5-HT1Dxcex2 (Weinshank et al., 1992a; Adham et al., 1992; Jin et al., 1992), 5-HT1Dxcex1 (Branchek et al. 1991; Hamblin and Metcalf, 1991; Weinshank et al., 1992a), 5-HT1E (Levy et al., 1992; McAllister et al., 1992, Zgombick et al., 1992) and 5-HT1F (Amlaiky et al., 1992; Adham et al., in press). These subtypes share  greater than 50% transmembrane amino acid identity and couple to the inhibition of adenylate cyclase. The 5-HT2 family includes the 5-HT2 receptor (Pritchett et al., 1988), 5-HT1C (Julius et al., 1988) and 5-HT2F (Rat Stomach Fundus; Foquet et al., 1992; Kursar et al., 1992). These receptors share approximately 70% amino acid identity and coupling to phosphoinositide hydrolysis. The 5-HT3 receptor has been shown to be a ligand-gated ion channel (Maricq et al., 1991). Heterogeneity of 5-HT3 receptors is controversial, although other ligand-gated ion channels display significant heterogeneity. Notably absent from this series are the 5-HT4 receptors. The second messenger coupling from tissue studies indicates activation of adenylate cyclase as a primary mode of signal transduction (Dumius et al., 1988; Bockaert et al., 1990). We report here the cloning of the first mammalian 5-HT receptor that couples to the stimulation of adenylate cyclase activity which we propose to name 5-HT4B. The pharmacological properties of this receptor indicate that it may be similar to a series of functionally defined 5-HT receptors described in the porcine vena cava (Trevethick et al., 1984), cat saphenous vein, coronary arteries (Cushing and Cohen, 1992), and several vascular dilatory effects (Mylecharane and Phillips, 1989). These receptors appear to underlie contractile and relaxant responses in isolated blood vessels indicating potential therapeutic benefit in angina, coronary artery disease, atherosclerosis, and possibly cerebral blood vessel disorders leading to stroke. The presence of this subtype in the CNS also indicates potential use in disorders of higher cognitive processes as well as control of autonomic function.
This invention provides an isolated nucleic acid molecule encoding a mammalian 5-HT4B receptor. In a preferred embodiment of this invention, the isolated nucleic acid encodes a human 5-HT4B receptor. In another embodiment of this invention, the nucleic acid molecule comprises a plasmid designated pcEXV-5-HT4B (ATCC Accession No. 75332).
This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a mammalian 5-HT4B receptor. This invention also provides a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding a human 5-HT4B receptor.
This invention provides an antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a mammalian 5-HT4B receptor so as to prevent translation of the mRNA molecule. This invention also provides an antisense oligonucleotide having a sequence capable of binding specifically to an mRNA molecule encoding a human 5-HT4B receptor so as to prevent translation of the mRNA molecule.
This invention provides a monoclonal antibody directed to a mammalian 5-HT4B receptor. This invention also provides a monoclonal antibody directed to a human 5-HT4B receptor.
This invention provides a pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a mammalian 5-HT4B receptor and a pharmaceutically acceptable carrier as well as a pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of mammalian 5-HT4B receptor and a pharmaceutically acceptable carrier.
This invention provides a pharmaceutical composition comprising an amount of a substance effective to alleviate the abnormalities resulting from overexpression of a human 5-HT4B receptor and a pharmaceutically acceptable carrier. This invention also provides pharmaceutical composition comprising an amount of a substance effective to alleviate abnormalities resulting from underexpression of a human 5-HT4B receptor and a pharmaceutically acceptable carrier.
This invention provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian 5-HT4B receptor so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA encoding the mammalian 5-HT4B receptor and when hybridized to mRNA encoding the mammalian 5-HT4B receptor, the complementary mRNA reduces the translation of the mRNA encoding the mammalian 5-HT4B receptor.
This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human 5-HT4B so positioned within such genome as to be transcribed into antisense mRNA complementary to mRNA encoding the human 5-HT4B and when hybridized to mRNA encoding the human 5-HT4B, the complementary mRNA reduces the translation of the mRNA encoding the human 5-HT4B.
This invention provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a mammalian 5-HT4B receptor positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the mammalian 5-HT4B receptor and when hybridized to mRNA encoding the 5-HT4B receptor, the antisense mRNA thereby prevents the translation of mRNA encoding the 5-HT4B receptor.
This invention also provides a transgenic, nonhuman mammal whose genome comprises DNA encoding a human 5-HT4B receptor positioned within such genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the human 5-HT4B receptor and when hybridized to mRNA encoding human 5-HT4B receptor, the antisense mRNA thereby prevents the translation of mRNA encoding the 5-HT4B receptor.
This invention also provides a method of determining the physiological effects of expressing varying levels of a mammalian 5-HT4B receptor which comprises producing a transgenic nonhuman animal whose levels of mammalian 5HT4B receptor expression are varied by use of an inducible promoter which regulates mammalian 5-HT4B receptor expression.
This invention also provides a method of determining the physiological effects of expressing varying levels of a human 5-HT4B receptor which comprises producing a transgenic nonhuman animal whose levels of human 5-HT4B receptor expression are varied by use of an inducible promoter which regulates human 5-HT4B receptor expression.
This invention further provides a method of determining the physiological effects of expressing varying levels of mammalian 5-HT4B receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of mammalian 5-HT4B receptor.
This invention further provides a method of determining the physiological effects of expressing varying levels of human 5-HT4B receptor which comprises producing a panel of transgenic nonhuman animals each expressing a different amount of human 5-HT4B receptor.
This invention provides a method for determining whether a compound not known to be capable of specifically binding to a human 5-HT4B receptor can specifically bind to the human 5-HT4B receptor, which comprises contacting a mammalian cell comprising a plasmid adapted for expression in a mammalian cell which plasmid further comprises DNA which expresses a human 5-HT4B receptor on the cell""s surface with the compound under conditions permitting binding of ligands known to bind to a human 5-HT4B receptor, detecting the presence of any compound bound to the human 5-HT4B receptor, the presence of bound ligand indicating that the compound is capable of specifically binding to the human 5-HT4B receptor.
This invention provides a method of screening compounds to identify drugs which interact with, and specifically bind to, a human 5-HT4B receptor on the surface of a cell, which comprises contacting a mammalian cell which comprises a plasmid adapted for expression in a mammalian cell which plasmid further comprises DNA which expresses a human 5-HT4B receptor on the cell""s surface with a plurality of compounds, determining those compounds which bind to the human 5-HT4B receptor expressed on the cell surface of the mammalian cell, and thereby identifying compounds which interact with, and specifically bind to, the human 5-HT4B receptor.
This invention provides a method for identifying a compound which is not known to be capable of binding to the human 5-HT4B receptor activates the human 5-HT4B receptor on the surface of a mammalian cell or prevents a ligand which does so, which comprises contacting the mammalian cell which cell comprises a plasmid adapted for expression in the mammalian cell such plasmid further comprising DNA which expresses the human 5-HT4B receptor on the cell surface of the mammalian cell with the compound under conditions permitting activation of or blockade of a functional response, determining whether the compound activates the human 5-HT4B receptor or prevents a ligand which does so, and thereby identifying the compound as a compound which interacts with, and activates the human 5-HT4B receptor or prevents the activation of the human 5-HT4B receptor by a ligand which does so.
This invention provides a method for diagnosing a predisposition to a disorder associated with the expression of a human 5-HT4B receptor allele which comprises: a.) obtaining DNA of subjects suffering from the disorder; b.) performing a restriction digest of the DNA with a panel of restriction enzymes; c.) electrophoretically separating the resulting DNA fragments on a sizing gel; d.) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing to DNA encoding a 5-HT4B receptor and labelled with a detectable marker; e.) detecting labelled bands which have hybridized to the DNA encoding a 5-HT4B receptor labelled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; f.) preparing DNA obtained for diagnosis by steps a-e; and g.) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step e and the DNA obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.